High confidence compound identification by liquid chromatography-mass spectrometry

ABSTRACT

Disclosed are methods for improving compound detection and characterization. Methods for characterizing a sample are disclosed. The methods can include providing a sample to a liquid chromatography system capable of sample separation to generate sample components; analyzing sample components by multiplexed targeted selected ion monitoring (SIM) to generate an inclusion list; and performing iterative mass spectral data-dependent acquisition (DDA) from the inclusion list, to identify individual sample components thereby characterizing the sample. In one example, multiplexed targeted SIMs and iterative MS2 DDA acquisition is used to increase robust compound identification for cell culture medium analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisional Application No. 62/968,525, filed Jan. 31, 2020, which isincorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention pertains to compound identification, and inparticular, to a method for high confidence compound identification byliquid chromatography-mass spectrometry (LC-MS), such as for antibodyprocess development.

BACKGROUND

Cell culture medium plays a key role in antibody production. It ishighly desirable and challenging to develop a deeper understanding ofhow individual components and their metabolites within the cell culturemedium impact the production performance. Despite the shift of usingchemically defined medium, soy-based medium is still widely used. Themajor components and impurities of soy hydrolysates have been shown toaffect antibodies' productivity and quality. Mass spectrometry playsimportant roles in compound quantification and identification in highlycomplex matrices. Liquid chromatography-mass spectrometry (LC-MS)-basedanalysis could benefit tremendously from improved data quality, whichcan subsequently lead to improved characterization with higherconfidence and less ambiguity.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of characterizinga sample, comprising: providing the sample to a liquid chromatographysystem capable of sample separation to generate sample components;analyzing sample components by multiplexed targeted selected ionmonitoring (SIM) to generate an inclusion list; and performing iterativemass spectral data-dependent acquisition (DDA) from the inclusion list,to identify individual sample components thereby characterizing thesample.

In some embodiments, the liquid chromatography system is a reversedphase liquid chromatography (RPLC) system.

In some embodiments, analyzing the ionized sample by multiplexedtargeted selected ion monitoring (SIM) to generate an inclusion listcomprises utilizing an ion trap or orbitrap mass analyzer.

In some embodiments, performing iterative mass spectral data-dependentacquisition (DDA) comprises utilizing an ion trap or orbitrap massanalyzer fitted with a segmented quadrupole mass filter.

In some embodiments, analyzing sample components by multiplexed targetedselected ion monitoring (SIM) to generate an inclusion list comprisessegmenting mass to ratio window settings in which multiple segments areincluded and each segment has multiple windows.

In some embodiments, multiple segments is three segments.

In some embodiments, multiple segments is four segments.

In some embodiments, multiple windows is 10 windows.

In some embodiments, each window within a segment has the same windowwidth.

In some embodiments, the sample is cell culture medium.

In some embodiments, the cell culture medium is a soy-based cell culturemedium.

In some embodiments, the cell culture medium is for a recombinantcell-based production system.

In some embodiments, the method is for characterizing components andtheir metabolites within the cell culture medium following incubationwith a recombinant cell-based production system.

In some embodiments, the recombinant cell-based production system is amammalian system.

In some embodiments, the recombinant cell-based production system is forprotein production.

In some embodiments, the protein is an antibody, a fusion protein,recombinant protein, or a combination thereof.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the monoclonal antibody is of isotype IgG1, IgG2,IgG3, IgG4, or mixed isotype.

Also disclosed is a method of compound identification for cell culturemedium analysis, comprising: providing the sample of cell culture mediumto a liquid chromatography system capable of sample separation togenerate sample components; analyzing sample components by multiplexedtargeted selected ion monitoring (SIM) to generate an inclusion list;and performing iterative mass spectral data-dependent acquisition (DDA)from the inclusion list, to identify individual compounds with the cellculture medium.

In some embodiments, the liquid chromatography system is a reversedphase liquid chromatography (RPLC) system.

In some embodiments, analyzing the fragmented sample by multiplexedtargeted selected ion monitoring (SIM) to generate an inclusion listcomprises utilizing an ion trap or orbitrap mass analyzer.

In some embodiments, performing iterative mass spectral data-dependentacquisition (DDA) comprises utilizing an ion trap or orbitrap massanalyzer fitted with a segmented quadrupole mass filter.

In some embodiments, analyzing sample components by multiplexed targetedselected ion monitoring (SIM) to generate an inclusion list comprisessegmenting mass to ratio window settings in which multiple segments areincluded and each segment has multiple windows.

In some embodiments, multiple segments is three segments.

In some embodiments, multiple segments is four segments.

In some embodiments, multiple windows is 10 windows.

In some embodiments, each window within a segment has the same windowwidth.

In some embodiments, the cell culture medium is a soy-based cell culturemedium.

In some embodiments, the cell culture medium is for a recombinantcell-based production system.

In some embodiments, the cell culture medium sample is a cell culturemedium sample obtained following incubation with a recombinantcell-based production system.

In some embodiments, the recombinant cell-based production system is amammalian system.

In some embodiments, the recombinant cell-based production system is forprotein production.

In some embodiments, the protein is an antibody, a fusion protein,recombinant protein, or a combination thereof.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the monoclonal antibody is of isotype IgG1, IgG2,IgG3, IgG4, or mixed isotype.

In various embodiments, any of the features or components of embodimentsdiscussed above or herein may be combined, and such combinations areencompassed within the scope of the present disclosure. Any specificvalue discussed above or herein may be combined with another relatedvalue discussed above or herein to recite a range with the valuesrepresenting the upper and lower ends of the range, and such ranges andall values falling within such ranges are encompassed within the scopeof the present disclosure. Each of the values discussed above or hereinmay be expressed with a variation of 1%, 5%, 10% or 20%. Otherembodiments will become apparent from a review of the ensuing detaileddescription.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic illustrating two types of MS1 data acquisition:(1) Conventional full-scan MS1 acquisition and (2) Targeted SIM MS1acquisition which is in accordance with embodiments disclosed herein. Asshown, targeted SIM MS1 acquisition allows for lower abundance speciesto be detected.

FIG. 2 shows a schematic illustrating an exemplary adjustable windowselection protocol.

FIG. 3 shows MS1 spectra acquired following either conventional fullscan MS1 acquisition (top tracing) or adjustable window SIMs MS1acquisition in accordance with embodiments disclosed herein (bottomtracing).

FIG. 4 shows a schematic illustrating two types of MS2 DDA: (1)Conventional DDA MS2 acquisition and (2) Iterative MS2 DDA platformacquisition in accordance with embodiments disclosed herein.

FIG. 5 shows iterative MS2 DDA results in more species being selectedand fragmented in comparison with conventional MS2 DDA where only highabundant species were selected and fragmented.

FIG. 6 shows a schematic comparing a conventional method ofcharacterizing a sample and using a combination of adjustable windowSIMs acquisition and iterative MS2 DDA in accordance with embodimentsdisclosed herein.

FIG. 7 shows a table illustrating the robustness and increasedsensitivity of adjustable Window SIMs acquisition as compared to fullscan MS. As the concentration of the compound decreased, the ability todetect the particular molecule was dependent upon the method utilized.Adjustable Window SIMs acquisition was capable of detecting molecules atlower concentrations than full scan MS.

FIG. 8 shows a graph illustrating the strength of iterative MS2 DDA. Thetop line represents the number of entries on the mass filter exclusionlist and bottom line is the number of entries on the mass filtersinclusion list each in relation to iterative injection. As illustrated,as the iterative injection number increases, the exclusion list ofspecies increases while the inclusion list decreases which in turnallows species of lower abundance to be fragmented and provide a robust,highly sensitive detection method.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims. Any embodiments or features of embodimentscan be combined with one another, and such combinations are expresslyencompassed within the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms “include,” “includes,” and “including,” aremeant to be non-limiting and are understood to mean “comprise,”“comprises,” and “comprising,” respectively.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Abbreviations Used Herein

ACN: Acetonitrile

CHO: Chinese Hamster Ovary

CQA: Critical Quality Attributes

CV: Coefficient of Variations

DDA: Data-Dependent Acquisition

EIC: Extracted Ion Chromatograph

ESI-MS: Electrospray Ionization Mass Spectrometry

FA: Formic Acid

HC: Heavy Chain

HILIC: Hydrophilic Interaction Liquid Chromatography

HMW: High Molecular Weight

IgG: Immunoglobulin G

IPA: Isopropanol

LC: Light Chain

LC-MS: Liquid Chromatography-Mass Spectrometry

LMW: Low Molecular Weight

mAb: Monoclonal Antibody

MS: Mass Spectrometry

MW: Molecular Weight

M/Z: Mass-to-Charge Ratio

NCE: Normalized Collision Energy

PK: Pharmacokinetics

PQA: Product Quality Attribute

PTM: Post-translational Modification

RP-LC: Reversed Phase Liquid Chromatography

SIM: Selected Ion Monitoring

Definitions

As used herein, the term “protein” includes any amino acid polymerhaving covalently linked amide bonds. Proteins comprise one or moreamino acid polymer chains, generally known in the art as “polypeptides.”“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. “Synthetic peptides orpolypeptides' refers to a non-naturally occurring peptide orpolypeptide. Synthetic peptides or polypeptides can be synthesized, forexample, using an automated polypeptide synthesizer. Various solid phasepeptide synthesis methods are known to those of skill in the art. Aprotein may contain one or multiple polypeptides to form a singlefunctioning biomolecule. A protein can include any of bio-therapeuticproteins, recombinant proteins used in research or therapy, trapproteins and other chimeric receptor Fc-fusion proteins, chimericproteins, antibodies, monoclonal antibodies, polyclonal antibodies,human antibodies, and bispecific antibodies. In another exemplaryaspect, a protein can include antibody fragments, nanobodies,recombinant antibody chimeras, cytokines, chemokines, peptide hormones,and the like. Proteins may be produced using recombinant cell-basedproduction systems, such as the insect bacculovirus system, yeastsystems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHOderivatives like CHO-K1 cells). For a recent review discussingbiotherapeutic proteins and their production, see Ghaderi et al.,“Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation,” (Biotechnol. Genet.Eng. Rev. (2012) 147-75). In some embodiments, proteins comprisemodifications, adducts, and other covalently linked moieties. Thosemodifications, adducts and moieties include for example avidin,streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose,neuraminic acid, N-acetylglucosamine, fucose, mannose, and othermonosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein(MBP), chitin binding protein (CBP), glutathione-S-transferase (GST)myc-epitope, fluorescent labels and other dyes, and the like. Proteinscan be classified on the basis of compositions and solubility and canthus include simple proteins, such as, globular proteins and fibrousproteins; conjugated proteins, such as, nucleoproteins, glycoproteins,mucoproteins, chromoproteins, phosphoproteins, metalloproteins, andlipoproteins; and derived proteins, such as, primary derived proteinsand secondary derived proteins.

Variant protein” or “protein variant”, or “variant” as used herein caninclude a protein that differs from a target protein by virtue of atleast one amino acid modification. Protein variant may refer to theprotein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about ten amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.The protein variant sequence herein will preferably possess at leastabout 80% homology with a parent protein sequence, and most preferablyat least about 90% homology, more preferably at least about 95%homology. In some exemplary embodiments, the protein can be an antibody,a bispecific antibody, a multispecific antibody, antibody fragment,monoclonal antibody, or combinations thereof.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds(i.e., “full antibody molecules”), as well as multimers thereof (e.g.IgM) or antigen-binding fragments thereof. Each heavy chain is comprisedof a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chainconstant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Invarious embodiments, the heavy chain may be an IgG isotype. In somecases, the heavy chain is selected from IgG1, IgG2, IgG3 or IgG4. Insome embodiments, the heavy chain is of isotype IgG1 or IgG4, optionallyincluding a chimeric hinge region of isotype IgG1/IgG2 or IgG4/IgG2.Each light chain is comprised of a light chain variable region (“LCVR or“V_(L)”) and a light chain constant region (CL). The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The term “antibody” includes reference toboth glycosylated and non-glycosylated immunoglobulins of any isotype orsubclass. The term “antibody” includes antibody molecules prepared,expressed, created or isolated by recombinant means, such as antibodiesisolated from a host cell transfected to express the antibody. For areview on antibody structure, see Lefranc et al., IMGT unique numberingfor immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains, 27(1) Dev. Comp. Immunol. 55-77 (2003); andM. Potter, Structural correlates of immunoglobulin diversity, 2(1) Surv.Immunol. Res. 27-42 (1983).

The term antibody also encompasses “bispecific antibody”, which includesa heterotetrameric immunoglobulin that can bind to more than onedifferent epitope. One half of the bispecific antibody, which includes asingle heavy chain and a single light chain and six CDRs, binds to oneantigen or epitope, and the other half of the antibody binds to adifferent antigen or epitope. In some cases, the bispecific antibody canbind the same antigen, but at different epitopes or non-overlappingepitopes. In some cases, both halves of the bispecific antibody haveidentical light chains while retaining dual specificity. Bispecificantibodies are described generally in U.S. Patent App. Pub. No.2010/0331527 (Dec. 30, 2010).

The term “antigen-binding portion” of an antibody (or “antibodyfragment”), refers to one or more fragments of an antibody that retainthe ability to specifically bind to an antigen. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature241:544-546), which consists of a VH domain, (vi) an isolated CDR, and(vii) an scFv, which consists of the two domains of the Fv fragment, VLand VH, joined by a synthetic linker to form a single protein chain inwhich the VL and VH regions pair to form monovalent molecules. Otherforms of single chain antibodies, such as diabodies are also encompassedunder the term “antibody” (see e.g., Holliger et al. (1993) 90 PNASU.S.A. 6444-6448; and Poljak et al. (1994) 2 Structure 1121-1123).

Moreover, antibodies and antigen-binding fragments thereof can beobtained using standard recombinant DNA techniques commonly known in theart (see Sambrook et al., 1989). Methods for generating human antibodiesin transgenic mice are also known in the art. For example, usingVELOCIMMUNE® technology (see, for example, U.S. Pat. No. 6,596,541,Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method forgenerating monoclonal antibodies, high affinity chimeric antibodies to adesired antigen are initially isolated having a human variable regionand a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody

The term “human antibody”, is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human mAbs of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo), for example in the CDRs and in particularCDR3. However, the term “human antibody”, as used herein, is notintended to include mAbs in which CDR sequences derived from thegermline of another mammalian species (e.g., mouse), have been graftedonto human FR sequences. The term includes antibodies recombinantlyproduced in a non-human mammal, or in cells of a non-human mammal. Theterm is not intended to include antibodies isolated from or generated ina human subject.

As used herein, the term “subject” refers to an animal, preferably amammal, more preferably a human, for example in need of amelioration,prevention and/or treatment of a disease or disorder.

As used herein, the term “impurity” can include any undesirable proteinpresent in the biopharmaceutical product. Impurity can include processand product-related impurities. The impurity can further be of knownstructure, partially characterized, or unidentified. Process-relatedimpurities can be derived from the manufacturing process and can includethe three major categories: cell substrate-derived, cell culture-derivedand downstream derived. Cell substrate-derived impurities include, butare not limited to, proteins derived from the host organism and nucleicacid (host cell genomic, vector, or total DNA). Cell culture-derivedimpurities include, but are not limited to, inducers, antibiotics,serum, and other media components. Downstream-derived impuritiesinclude, but are not limited to, enzymes, chemical and biochemicalprocessing reagents (e.g., cyanogen bromide, guanidine, oxidizing andreducing agents), inorganic salts (e.g., heavy metals, arsenic,nonmetallic ion), solvents, carriers, ligands (e.g., monoclonalantibodies), and other leachables. Product-related impurities (e.g.,precursors, certain degradation products) can be molecular variantsarising during manufacture and/or storage that do not have propertiescomparable to those of the desired product with respect to activity,efficacy, and safety. Such variants may need considerable effort inisolation and characterization in order to identify the type ofmodification(s). Product-related impurities can include truncated forms,modified forms, and aggregates. Truncated forms are formed by hydrolyticenzymes or chemicals which catalyze the cleavage of peptide bonds.Modified forms include, but are not limited to, deamidated, isomerized,mismatched S—S linked, oxidized, or altered conjugated forms (e.g.,glycosylation, phosphorylation). Modified forms can also include anypost-translational modification form. Aggregates include dimers andhigher multiples of the desired product (Q6B Specifications: TestProcedures and Acceptance Criteria for Biotechnological/BiologicalProducts, ICH August 1999, U.S. Dept. of Health and Humans Services).

The term “low molecular weight (LMVV) protein drug impurity” includesbut is not limited to precursors, degradation products, truncatedspecies, proteolytic fragments including Fab fragments, Fc or heavychain fragments, ligand or receptor fragments, H2L (2 heavy chains and 1light chain), H2 (2 heavy chains), HL (1 heavy chain and 1 light chain),HC (1 heavy chain), and LC (1 light chain) species. A LMW protein drugimpurity can be any variant which is an incomplete version of theprotein product, such as one or more components of a multimeric protein.Protein drug impurity, drug impurity or product impurity are terms thatmay be used interchangeably throughout the specification. LMW drug orproduct impurities are generally considered molecular variants withproperties such as activity, efficacy, and safety that may be differentfrom those of the desired drug product.

Degradation of protein product is problematic during production of theprotein drug product in cell culture systems. For example, proteolysisof a protein product may occur due to release of proteases in cellculture medium. Medium additives, such as soluble iron sources added toinhibit metalloproteases, or serine and cysteine proteases inhibitors,have been implemented in cell culture to prevent degradation (Clincke,M.-F., et al, BMC Proc. 2011, 5, P115). C-terminal fragments may becleaved during production due to carboxyl peptidases in the cell culture(Dick, L W et al, Biotechnol Bioeng 2008; 100:1132-43).

The term “high molecular weight (HMW) protein drug impurity” includesbut is not limited to mAb trimers and mAb dimers. HMW species can bedivided into two groups: 1) monomer with extra light chains (H2L3 andH2L4 species) and 2) monomer plus Fab fragments complexes. In addition,after treatment with IdeS enzymatic digestion, different dimerizedfragments (Fab2-Fab2, Fc-Fc and Fab2-Fc) are formed.

A “post-translational modification” (PTM) refers to the covalentmodification of proteins following protein biosynthesis.Post-translational modifications can occur on the amino acid side chainsor at the protein's C- or N-termini. PTMs are generally introduced byspecific enzymes or enzyme pathways. Many occur at the site of aspecific characteristic protein sequence (e.g., signature sequence)within the protein backbone. Several hundred PTMs have been recorded,and these modifications invariably influence some aspect of a protein'sstructure or function (Walsh, G. “Proteins” (2014) second edition,published by Wiley and Sons, Ltd., ISBN: 9780470669853). The variouspost-translational modifications include, but are not limited to,cleavage, N-terminal extensions, protein degradation, acylation of theN-terminus, biotinylation (acylation of lysine residues with a biotin),amidation of the C-terminal, glycosylation, iodination, covalentattachment of prosthetic groups, acetylation (the addition of an acetylgroup, usually at the N-terminus of the protein), alkylation (theaddition of an alkyl group (e.g. methyl, ethyl, propyl) usually atlysine or arginine residues), methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications(proline and lysine hydroxylations and carboxy terminal amidation),Vitamin K dependent modification wherein Vitamin K is a cofactor in thecarboxylation of glutamic acid residues resulting in the formation of aγ-carboxyglutamate (a glu residue), glutamylation (covalent linkage ofglutamic acid residues), glycylation (covalent linkage glycineresidues), glycosylation (addition of a glycosyl group to eitherasparagine, hydroxylysine, serine, or threonine, resulting in aglycoprotein), isoprenylation (addition of an isoprenoid group such asfarnesol and geranylgeraniol), lipoylation (attachment of a lipoatefunctionality), phosphopantetheinylation (addition of a4′-phosphopantetheinyl moiety from coenzyme A, as in fatty acid,polyketide, non-ribosomal peptide and leucine biosynthesis),phosphorylation (addition of a phosphate group, usually to serine,tyrosine, threonine or histidine), and sulfation (addition of a sulfategroup, usually to a tyrosine residue). The post-translationalmodifications that change the chemical nature of amino acids include,but are not limited to, citrullination (e.g., the conversion of arginineto citrulline by deimination), and deamidation (e.g., the conversion ofglutamine to glutamic acid or asparagine to aspartic acid). Thepost-translational modifications that involve structural changesinclude, but are not limited to, formation of disulfide bridges(covalent linkage of two cysteine amino acids) and proteolytic cleavage(cleavage of a protein at a peptide bond). Certain post-translationalmodifications involve the addition of other proteins or peptides, suchas ISGylation (covalent linkage to the ISG15 protein(Interferon-Stimulated Gene)), SUMOylation (covalent linkage to the SUMOprotein (Small Ubiquitin-related MOdifier)) and ubiquitination (covalentlinkage to the protein ubiquitin). See www.uniprot.org/docs/ptmlist fora more detailed controlled vocabulary of PTMs curated by UniProt.

The term as used herein, “glycopeptide/glycoprotein” is a modifiedpeptide/protein, during or after their synthesis, with covalently bondedcarbohydrates or glycan. In certain embodiments, a glycopeptide isobtained from a monoclonal antibody, for example, from a protease digestof a monoclonal antibody.

The term as used herein, “glycan” is a compound comprising one or moreof sugar units which commonly include glucose (Glc), galactose (Gal),mannose (Man), fucose (Fuc), N-acetylgalactosamine (GaINAc),N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuNAc) (FrankKjeldsen, et al. Anal. Chem. 2003, 75, 2355-2361). The glycan moiety inglycoprotein, such as a monoclonal antibody, is an important characterto identify its function or cellular location. For example, a specificmonoclonal antibody is modified with specific glycan moiety.

The term “sample,” as used herein, refers to a mixture of molecules suchcomponents within cell culture medium that is subjected to manipulationin accordance with the methods of the invention, including, for example,separating, analyzing, or profiling. A sample can comprise at least ananalyte molecule, e.g., glycopeptide, such as obtained from a monoclonalantibody, that is subjected to manipulation in accordance with themethods of the invention, including, for example, separating, analyzing,extracting, concentrating or profiling.

The terms “analysis” or “analyzing,” as used herein, are usedinterchangeably and refer to any of the various methods of separating,detecting, isolating, purifying, solubilizing, detecting and/orcharacterizing molecules of interest. Examples include, but are notlimited to, solid phase extraction, solid phase micro extraction,electrophoresis, mass spectrometry, e.g., Multiplexed targeted selectedion monitoring (SIM)-MS followed by iterative MS2 DDA, ESI-MS, SPEHILIC, or MALDI-MS, liquid chromatography, e.g., high performance, e.g.,reverse phase, normal phase, or size exclusion, ion-pair liquidchromatography, liquid-liquid extraction, e.g., accelerated fluidextraction, supercritical fluid extraction, microwave-assistedextraction, membrane extraction, soxhlet extraction, precipitation,clarification, electrochemical detection, staining, elemental analysis,Edmund degradation, nuclear magnetic resonance, infrared analysis, flowinjection analysis, capillary electrochromatography, ultravioletdetection, and combinations thereof.

The term “profiling,” as used herein, refers to any of various methodsof analysis which are used in combination to provide the content,composition, or characteristic ratio of compounds, such as proteins.

“Contacting,” as used herein, includes bringing together at least twosubstances in solution or solid phase.

“Peptide mapping analysis” as used herein includes experiments whereinthe protein undergoes digestion followed by separation of the resultingpeptides and their analysis, preferably using LC-MS. In some exemplaryembodiments, peptide mapping analysis can be applied to confirm theprimary sequence of protein biopharmaceuticals, where a protein moleculecan be first hydrolyzed into small peptide fragments using a hydrolyzingagent and then the amino acid sequence of each peptide fragment isdetermined by LC-MS analysis taking into consideration of the cDNApredicted sequence and the specificity of the protease used. Data frompeptide mapping analysis could also be utilized to identify and quantifypost-translational modifications, confirm the disulfide bond linkagesand even detect amino acid substitution events present at very lowlevels (<0.1%) (Zeck et al. PloS one 2012, 7, e40328). During peptidemapping analysis of protein biopharmaceuticals, LC-MS can be oftenperformed in combination with ultraviolet (UV) detection to generateso-called UV fingerprints, which alone can be used as an identificationassay during quality control (QC) and drug release.

As used herein, the term “digestion” refers to hydrolysis of one or morepeptide bonds of a protein. There are several approaches to carrying outdigestion of a protein in a sample using an appropriate hydrolyzingagent, for example, enzymatic digestion or non-enzymatic digestion. Asused herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different agents that can performdigestion of a protein. Non-limiting examples of hydrolyzing agents thatcan carry out enzymatic digestion include trypsin, endoproteinase Arg-C,endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T(OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes(IdeS), chymotrypsin, pepsin, thermolysin, papain, pronase, and proteasefrom Aspergillus saitoi. Non-limiting examples of hydrolyzing agentsthat can carry out non-enzymatic digestion include the use of hightemperature, microwave, ultrasound, high pressure, infrared, solvents(non-limiting examples are ethanol and acetonitrile), immobilized enzymedigestion (IMER), magnetic particle immobilized enzymes, and on-chipimmobilized enzymes. For a recent review discussing the availabletechniques for protein digestion see Switazar et al., “ProteinDigestion: An Overview of the Available Techniques and RecentDevelopments” (J. Proteome Research 2013, 12, 1067-1077). One or acombination of hydrolyzing agents can cleave peptide bonds in a proteinor polypeptide, in a sequence-specific manner, generating a predictablecollection of shorter peptides.

Several approaches are available that can be used to digest a protein.One of the widely accepted methods for digestion of proteins in a sampleinvolves the use of proteases. Many proteases are available and each ofthem has their own characteristics in terms of specificity, efficiency,and optimum digestion conditions. Proteases refer to both endopeptidasesand exopeptidases, as classified based on the ability of the protease tocleave at non-terminal or terminal amino acids within a peptide.Alternatively, proteases also refer to the six distinctclasses—aspartic, glutamic, and metalloproteases, cysteine, serine, andthreonine proteases, as classified on the mechanism of catalysis. Theterms “protease” and “peptidase” are used interchangeably to refer toenzymes which hydrolyze peptide bonds. Proteases can also be classifiedinto specific and non-specific proteases. As used herein, the term“specific protease” refers to a protease with an ability to cleave thepeptide substrate at a specific amino acid side chain of a peptide. Asused herein, the term “non-specific protease” refers to a protease witha reduced ability to cleave the peptide substrate at a specific aminoacid side chain of a peptide. A cleavage preference may be determinedbased on the ratio of the number of a particular amino acid as the siteof cleavage to the total number of cleaved amino acids in the proteinsequences.

The protein can optionally be prepared before characterizing. In someexemplary embodiments, the protein preparation includes a step ofprotein digestion. In some specific exemplary embodiments, the proteinpreparation includes a step of protein digestion, wherein the proteindigestion can be carried out using trypsin.

In some exemplary embodiments, the protein preparation can include astep for denaturing the protein, reducing the protein, buffering theprotein, and/or desalting the sample, before the step of proteindigestion. These steps can be accomplished in any suitable manner asdesired.

To provide characterization of different protein attributes using eitherpeptide mapping analysis or intact mass analysis, a wide variety ofLC-MS based assays can be performed.

As used herein, the term “liquid chromatography” refers to a process inwhich a chemical mixture carried by a liquid can be separated intocomponents as a result of differential distribution of the chemicalentities as they flow around or over a stationary liquid or solid phase.Non-limiting examples of liquid chromatography include reverse phaseliquid chromatography, ion-exchange chromatography, size exclusionchromatography, affinity chromatography, and hydrophobic chromatography.

As used herein, the term “mass spectrometer” refers to a device capableof detecting specific molecular species and accurately measuring theirmasses. The term can be meant to include any molecular detector intowhich a polypeptide or peptide may be eluted for detection and/orcharacterization. A mass spectrometer consists of three major parts: theion source, the mass analyzer, and the detector. The role of the ionsource is to create gas phase ions. Analyte atoms, molecules, orclusters can be transferred into gas phase and ionized eitherconcurrently (as in electrospray ionization). The choice of ion sourcedepends on the application.

As used herein, “mass analyzer” refers to a device that can separatespecies, that is, atoms, molecules, or clusters, according to theirmass. Non-limiting examples of mass analyzers that could be employed aretime-of-flight (TOF), magnetic/electric sector, quadrupole mass filter(Q), quadrupole ion trap (QIT), orbitrap, Fourier transform ioncyclotron resonance (FTICR), and also the technique of accelerator massspectrometry (AMS).

As used herein, “mass-to-charge ratio” or “m/z” is used to denote thedimensionless quantity formed by dividing the mass of an ion in unifiedatomic mass units by its charge number (regardless of sign). In general,the charge state depends on: the method of ionization (as electrosprayionization, ESI tends to promote multiple ionization, which is not asfrequent in MALDI), peptide length (as longer peptides have more groupswhere additional protons can be attached (basic residues)), peptidesequence (as some amino acids (e.g., Arg or Lys) are more susceptible toionization than others), the instrument settings, solvent pH, andsolvent composition.

As used herein, the term “tandem mass spectrometry” refers to atechnique where structural information on sample molecules can beobtained by using multiple stages of mass selection and mass separation.A prerequisite is that the sample molecules can be transferred into gasphase and ionized intact and that they can be induced to fall apart insome predictable and controllable fashion after the first mass selectionstep. Multistage MS/MS, or MSn, can be performed by first selecting andisolating a precursor ion (MS2), fragmenting it, isolating a primaryfragment ion (MS3), fragmenting it, isolating a secondary fragment(MS4), and so on as long as one can obtain meaningful information or thefragment ion signal is detectable. Tandem MS have been successfullyperformed with a wide variety of analyzer combinations. What analyzersto combine for a certain application can be determined by many differentfactors, such as sensitivity, selectivity, and speed, but also size,cost, and availability. The two major categories of tandem MS methodsare tandem-in-space and tandem-in-time, but there are also hybrids wheretandem-in-time analyzers are coupled in space or with tandem-in-spaceanalyzers.

A tandem-in-space mass spectrometer comprise of an ion source, aprecursor ion activation device, and at least two non-trapping massanalyzers. Specific m/z separation functions can be designed so that inone section of the instrument ions are selected, dissociated in anintermediate region, and the product ions are then transmitted toanother analyzer for m/z separation and data acquisition.

In tandem-in-time mass spectrometer ions produced in the ion source canbe trapped, isolated, fragmented, and m/z separated in the same physicaldevice.

“Targeted mass spectrometry,” as used herein, is a mass spectrometrytechnique that uses multiple stages of tandem mass spectrometry (MSnwith n=2 or 3) for ions of specific mass (m/z), at specific time. Thevalues of the m/z and time are defined in an inclusion list which isderived from a previous analysis.

As used herein, the term “quadrupole-Orbitrap hybrid mass spectrometer”refers to a hybrid system made by coupling a quadrupole massspectrometer to an orbitrap mass analyzer. A tandem in-time experimentusing the quadrupole-Orbitrap hybrid mass spectrometer begins withejection of all ions except those within a selected, narrow m/z rangefrom the quadrupole mass spectrometer. The selected ions can be insertedinto orbitrap and fragmented most often by low-energy CID. Fragmentswithin the m/z acceptance range of the trap should remain in the trap,and an MS-MS spectrum can be obtained. Similar hybrid systems can beused for fast protein sequencing, such as, but not limited to QIT-FTICRand Qq-FTICR.

As used herein, the term “protein de novo sequencing” refers to aprocedure for determination of the amino acid sequence of a peptidewithout relying on the information gained from other sources. Due to thehigh level of sensitivity of mass spectrometry, this technique canprovide vital information that is often beyond the capabilities ofconventional sequencing methods.

As used herein, the term “protein sequence coverage” refers to thepercentage of the protein sequence covered by identified peptides. Thepercent coverage can be calculated by dividing the number of amino acidsin all found peptides by the total number of amino acids in the entireprotein sequence.

As used herein, the term “database” refers to bioinformatic tools whichprovide the possibility of searching the uninterpreted MS-MS spectraagainst all possible sequences in the database(s). Non-limiting examplesof such tools are Mascot (www.matrixscience.com), Spectrum Mill(www.chem.agilent.com), PLGS (www.waters.com), PEAKS(www.bioinformaticssolutions.com), Proteinpilot(download.appliedbiosystems.com//proteinpilot), Phenyx(www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA(www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem(www.thegpm.org/TANDEM/), Protein Prospector(www.prospector.ucsf.edu/prospector/mshome.htm), Byonic(www.proteinmetrics.com/products/byonic) or Sequest(fields.scripps.edu/sequest).

General Description

From the foregoing it will be appreciated that a need exists forimproved methods and systems to improve compound detection andcharacterization. The disclosed invention meets that need. Disclosedherein are methods for high confidence compound identification by liquidchromatography-mass spectrometry (LC-MC), such as for antibody processdevelopment. Embodiments disclosed herein provide methods for robust,highly sensitivity sample characterization by combining multiplexedtargeted SIMs and iterative MS2 DDA acquisition.

In some exemplary embodiments, the method includes providing a sample toa liquid chromatography system capable of sample separation to generatesample components; analyzing sample components by multiplexed targetedselected ion monitoring (SIM) to generate an inclusion list; andperforming iterative mass spectral data-dependent acquisition (DDA) fromthe inclusion list, to identify individual sample components therebycharacterizing the sample.

In some embodiments, providing the sample to a liquid chromatographysystem capable of sample separation to generate sample componentscomprises providing the sample to a reverse phase liquid chromatography(RPLC) system, ion-exchange chromatography system, size exclusionchromatography system, affinity chromatography system,hydrophilic-interaction chromatography system, or hydrophobicchromatography system.

In some embodiments, the liquid chromatography system is a RPLC system.In some particular embodiments, RPLC analysis is performed using aSupelco Discovery HS F5-3 column. The column temperature can bemaintained at a constant temperature throughout the chromatography run,e.g., using a commercial column heater. In some embodiments, the columnis maintained at a temperature between about 18° C. to about 70° C.,e.g., about 30° C. to about 60° C., about 40° C. to about 50° C., e.g.,at about 20° C., about 25° C., about 30° C., about 35° C., about 40° C.,about 45° C., about 50° C., about 55° C., about 60° C., about 65° C., orabout 70° C. In some embodiments, the column temperature is about 40° C.In some embodiments, the run time can be between about 15 to about 240minutes, e.g., about 20 to about 70 min, about 30 to about 60 min, about40 to about 90 min, about 50 min to about 100 min, about 60 to about 120min, about 50 to about 80 min.

In some embodiments, the mobile phase is an aqueous mobile phase. Arepresentative aqueous mobile phase contains 140 mM sodium acetate and10 mM ammonium bicarbonate. The UV traces are typically recorded at 215and 280 nm.

In some exemplary embodiments, the mobile phase used to elute theprotein can be a mobile phase that can be compatible with a massspectrometer.

In some exemplary embodiments, the mobile phase used in the liquidchromatography device can include water, acetonitrile, trifluoroaceticacid, formic acid, or combination thereof.

In some exemplary embodiments, the mobile phase can have a flow rate ofabout 0.1 ml/min to about 0.4 ml/min in the liquid chromatographydevice. In one aspect, the flow rate of the mobile phase in the liquidchromatography device can be about 0.1 ml/min, about 0.15 ml/min, about0.20 ml/min, about 0.25 ml/min, about 0.30 ml/min, about 0.35 ml/min, orabout 0.4 ml/min.

In some embodiments, analyzing sample components by multiplexed targetedselected ion monitoring (SIM) to generate an inclusion list comprisesutilizing an ion trap or orbitrap mass analyzer. In some embodiments,the ion trap or orbitrap mass analyzer is a Thermo Q Exactive HFOrbitrap mass spectrometer.

In some embodiments, analyzing sample components by multiplexed targetedselected ion monitoring (SIM) to generate an inclusion list comprisessegmenting mass to ratio window settings in which multiple segments areincluded and each segment has multiple windows. In some embodiments, atleast two, at least three, at least four, at least five or more segmentsare used. In some embodiments, three segments are used. In someembodiments, four segments are used.

In some embodiments, each segment includes multiple windows of the samewidth. In some embodiments, at least 2 or more windows are used, such asat least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10 windows are used. In some embodiments, eachsegment includes 10 windows.

In some embodiments, window width varies amongst the multiple segments.For example, the window width in ranges of m/z of most interest are morenarrow than those of less interest. In some embodiments, the segmentsone and two have window widths of a narrower width than segments threeand four. For example, as shown in FIG. 2, an exemplary regiment caninclude four segments ranging from 60 to 760.5 m/z: Segment 1, 60 m/z to155.5 m/z with a window width of 10 Da; Segment 2, 155 m/z to 250.5 m/z,window width 10 Da; Segment 3, 250 m/z to 505.5 m/z, window width 25 DA;and Segment 4, 505.5 m/z to 760.5 m/z, window width 25 Da wherein eachsegment includes 10 windows and 0.5 Da overlap between adjacent windows.

In embodiments, following generation of an inclusion list the methodincludes performing iterative MS2 DDA from the inclusion list toidentify individual sample components thereby characterizing the sample.

In some exemplary embodiments, iterative MS DDA utilizes an ion trap ororbitrap mass analyzer fitted with a segmented quadrupole mass filter.

In some exemplary embodiments, iterative MS DDA utilizes a commerciallyavailable ion trap or orbitrap mass analyzer fitted with a segmentedquadrupole mass filter, such as a Thermo Orbitrap™ Fusion Lumos massspectrometer.

In some exemplary embodiments, data is processed following iterative MSDDA, such as by use of

In some embodiments, the sample is cell culture medium, such as cellculture medium used in fed-batch cell culture, continuous cell cultureor perfusion cell culture.

In some embodiments, the sample cell culture medium is a soy-based cellculture medium.

In some exemplary embodiments, the cell culture medium is for arecombinant cell-based production system, such as a mammalian system. Insome embodiments,

In some embodiments, the method is for characterizing components andtheir metabolites within the cell culture medium prior or followingincubation with a recombinant cell-based production system. In someembodiments, the recombinant cell-based production system is for proteinproduction. Exemplary proteins include, but are not limited to, anantibody, a fusion protein, recombinant protein, or a combinationthereof.

In some embodiments, the antibody is a bispecific antibody, antibodyfragment or a multispecific antibody.

In some exemplary embodiments, the antibody is a monoclonal antibody,such as, but not limited to, a monoclonal antibody of isotype IgG1,IgG2, IgG3, IgG4, or mixed isotype.

In some exemplary embodiments, the protein is be a therapeutic protein.

In some exemplary embodiments, the protein can be an immunoglobulinprotein.

In one exemplary embodiment, the protein can be a protein variant.

In one exemplary embodiment, the protein can be a post-translationallymodified protein.

In one exemplary embodiment, the post-translationally modified proteincan be a formed by cleavage, N-terminal extensions, protein degradation,acylation of the N-terminus, biotinylation, amidation of the C-terminal,oxidation, glycosylation, iodination, covalent attachment of prostheticgroups, acetylation, alkylation, methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications,Vitamin K dependent modification, glutamylation, glycylation,glycosylation, deglycosylation, isoprenylation, lipoylation,phosphopantetheinylation, phosphorylation, sulfation, citrullination,deamidation, formation of disulfide bridges, proteolytic cleavage,ISGylation, SUMOylation or ubiquitination (covalent linkage to theprotein ubiquitin).

In one exemplary embodiment, the post-translationally modified proteincan be formed on oxidation of a protein.

In another exemplary embodiment, the cell culture medium can include adegradation product, such as a post-translation modification of atherapeutic protein.

In some exemplary embodiments, the protein can be a protein with a pl inthe range of about 4.5 to about 9.0. In one aspect, the protein can be aprotein with a pl of about 4.5, about 5.0, about 5.5, about 5.6, about5.7, about 5.8, about 5.9, about 6.0, about 6.1 about 6.2, about 6.3,about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about 7.6,about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 about 8.2, about8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9,or about 9.0.

In some embodiments, the identified components include, but are notlimited to, amino acids, dipeptides, tripeptides, or a combinationthereof with an abundance dynamic range of at least 2 orders ofmagnitude, such as at least 3 orders of magnitude, at least 4 orders ofmagnitude or greater. In some embodiments, the identified componentsinclude one or more of the compounds provided in FIG. 7.

In some exemplary embodiments, one or more compounds/components detectedcan include one or more product-related impurities. Exemplary productrelated impurities can be, but are not limited to, molecular variants,precursors, degradation products, fragmented protein, digested product,aggregates, post-translational modification form or combinationsthereof.

In some specific exemplary embodiments, the one or morecompounds/components detected can be a process-related impurity. Theprocess-related impurity can include impurities derived from themanufacturing process, e.g., nucleic acids and host cell proteins,antibiotics, serum, other media components, enzymes, chemical andbiochemical processing reagents, inorganic salts, solvents, carriers,ligands, and other leachables used in the manufacturing process that maybe present or released into the cell culture medium.

In some embodiments, the disclosed method is a method of compoundidentification for cell culture medium analysis, comprising: providingthe sample of cell culture medium to a liquid chromatography systemcapable of sample separation to generate sample components; analyzingsample components by multiplexed targeted selected ion monitoring (SIM)to generate an inclusion list; and performing iterative mass spectraldata-dependent acquisition (DDA) from the inclusion list, to identifyindividual compounds within the cell culture medium. In some examples,the method further includes culturing cells producing a recombinantprotein, such as antibody, for example in cell culture medium, obtaininga sample from the cell culture at a desired time point prior tocharacterizing the cell culture medium components.

In some embodiments, the method includes identifying cell culture mediumcomponents by the using the disclosed method followed by modifying oneor more culture conditions of the cell culture to reduce the amount ofcharacterized compound produced during cell culture of the protein.Exemplary conditions of the cell culture that can be changed include,but are not limited to, temperature, pH, cell density, amino acidconcentration, osmolality, growth factor concentration, agitation, gaspartial pressure, surfactants, or combinations thereof.

In some embodiments, the cells producing the protein, such as theantibody, are CHO cells. In other embodiments, the cells are hybridomacells.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods of the invention, and are not intended to limitthe scope of what the inventors regard as their invention. Efforts havebeen made to ensure accuracy with respect to numbers used (e.g.,amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is average molecular weight, temperature is indegrees Centigrade, room temperature is about 25° C., and pressure is ator near atmospheric.

Example 1: Materials and Methods

Pooled soy hydrolysates were dissolved in Milli-Q water as the matrix.Stable isotope labeled compound standards were spiked in with finalconcentration ranging from several nanomolar to hundreds of millimolar.A Supelco Discovery HS F5-3 column was used for reversed-phase liquidchromatography (RPLC) separation. Multiplexed targeted selected ionmonitoring (SIM) was performed on a Thermo Q Exactive HF Orbitrap massspectrometer to generate the inclusion list. Iterative MS2data-dependent acquisition (DDA) from inclusion list was conducted on aThermo Orbitrap Fusion Lumos mass spectrometer. MS1 full scan followedby conventional MS2 DDA was performed as comparison. Compound Discoverer3.1 was used for data processing.

Example 2: Multiplexed Targeted SIMs and Iterative MS2 DDA AcquisitionIncrease Robust Compound Identification for Cell Culture Medium Analysis

Using the materials and methods described in Example 1, multiplexedtargeted SIMs and iterative MS2 DDA acquisition was performed. FIG. 1provides a schematic illustrating two types of MS1 data acquisition: (1)Conventional full-scan MS1 acquisition and (2) Targeted SIM MS1acquisition which is in accordance with embodiments disclosed herein.Targeted SIM MS1 acquisition relies upon sorting molecules based uponsize. By comparison, full scan MS1 detects a particular number ofmolecules regardless of size. As shown, targeted SIM MS1 acquisitionallows for lower abundance species to be detected as only a particularnumber of each size of molecules is detected, thereby improving theconfidence of detection. Targeted SIM MS1 included optimizing injectiontime and isolation windows.

In particular, FIG. 2 provides a schematic illustrating an exemplaryadjustable window selection regimen which includes four segments rangingfrom 60 to 760.5 m/z: Segment 1, 60 m/z to 155.5 m/z with a window widthof 10 Da; Segment 2, 155 m/z to 250.5 m/z, window width 10 Da; Segment3, 250 m/z to 505.5 m/z, window width 25 DA; and Segment 4, 505.5 m/z to760.5 m/z, window width 25 Da. Each segment included 10 windows and 0.5Da overlap between adjacent windows. The width of the window variedbetween segments, however, it did not differ within a particularsegment. Regions of particular interest had a smaller window width. FIG.3 shows MS1 spectrums acquired following either conventional full scanMS1 acquisition (top tracing) or adjustable window SIMs MS1 acquisitionin accordance with embodiments disclosed herein (bottom tracing).Comparing to MS1 full scan, multiplexed targeted SIMs provided cleanerbackground and higher intensity for several spiked-in stable isotopelabeled compounds. For example, 10 nM of stable isotope labeledhistidine was only observed by multiplexed targeted SIMs method.

Following MS1 full scan or multiplexed targeted SIM, MS2 DDA wasperformed. FIG. 4 shows a schematic illustrating two types of MS2 DDA:(1) Conventional DDA MS2 acquisition and (2) Iterative DDA MS2 platformin accordance with embodiments disclosed herein.

FIG. 5 shows iterative MS2 data acquisition results in more speciesbeing selected and fragmented in comparison with conventional DDA MS2acquisition where only high abundant species were selected andfragmented. FIG. 6 shows a schematic comparing a conventional method ofcharacterizing a sample and using a combination of adjustable windowSIMs acquisition and iterative MS2 acquisition in accordance withembodiments disclosed herein. As shown in FIG. 7, the combination ofadjustable window SIMs acquisition and Iterative MS2 acquisitionidentified a variety of components with great complexity in soyhydrolysate samples, including amino acids, dipeptides, tripeptides,etc. with the abundance dynamic range of at least 4 orders of magnitude.FIG. 8 shows a graph illustrating the strength of iterative MS2acquisition. The top line represents the number of entries on the massfilter exclusion list and bottom line is the number of entries on themass filters inclusion list each in relation to iterative injection. Asillustrated, as iterative injection increases, the exclusion list ofspecies increases while the inclusion list decreases which in turnallows species of lower abundance to be detected. In the iterative MS2DDA, the background ions in the blank run were successfully excludedduring sample runs. With multiple iterative sample runs, MS2 scans weretriggered on much lower abundant ions. Fast and high confidencecomponent identification was achieved by searching against in-houselibrary containing accurate mass, retention time and fragmentationspectra besides other online databases. It is contemplated that thedisclosed method can be applied to analyze cell metabolites, such as forCHO cell metabolites analysis during cell culture development.

Overall, the disclosed methods provide a more robust, sensitive methodfor characterizing samples, such as cell culture medium and individualcomponents and metabolites therein, which can be used to improveantibody process development.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method of characterizing a sample, comprising:providing the sample to a liquid chromatography system capable of sampleseparation to generate sample components; analyzing sample components bymultiplexed targeted selected ion monitoring (SIM) to generate aninclusion list; and performing iterative mass spectral data-dependentacquisition (DDA) from the inclusion list, to identify individual samplecomponents.
 2. The method of claim 1, wherein the liquid chromatographysystem is a reversed phase liquid chromatography (RPLC) system.
 3. Themethod of claim 1, wherein analyzing the sample components bymultiplexed targeted selected ion monitoring (SIM) to generate aninclusion list comprises utilizing an ion trap or orbitrap massanalyzer; and wherein performing iterative mass spectral DDA comprisesutilizing the ion trap or orbitrap mass analyzer fitted with a segmentedquadrupole mass filter.
 4. The method of claim 1, wherein analyzingsample components by multiplexed targeted selected ion monitoring (SIM)to generate an inclusion list comprises segmenting mass to ratio windowsettings in which multiple segments are included and each segment hasmultiple windows.
 5. The method of claim 4, wherein multiple segments isthree segments or four segments; and wherein multiple windows is 10windows.
 6. The method of claim 4, wherein each window within a segmenthas the same window width.
 7. The method of claim 1, wherein the sampleis cell culture medium.
 8. The method of claim 7, wherein the method isfor characterizing components and their metabolites within the cellculture medium; and wherein the cell culture medium is a soy-based cellculture medium and/or wherein the cell culture medium is for arecombinant cell-based production system.
 9. The method of claim 7,wherein the method is for characterizing components and theirmetabolites within the cell culture medium following incubation with arecombinant cell-based production system.
 10. The method of claim 9,wherein the recombinant cell-based production system is a mammaliansystem for protein production; and wherein the protein is an antibody, afusion protein, recombinant protein, or a combination thereof.
 11. Themethod of claim 10, wherein the antibody is a monoclonal antibody ofisotype IgG1, IgG2, IgG3, IgG4, or mixed isotype.
 12. A method ofcompound identification for cell culture medium analysis, comprising:providing the sample of cell culture medium to a reversed phase liquidchromatography system (RPLC) capable of sample separation to generatesample components; analyzing sample components by multiplexed targetedselected ion monitoring (SIM) to generate an inclusion list; andperforming iterative mass spectral data-dependent acquisition (DDA) fromthe inclusion list, to identify individual compounds with the cellculture medium.
 13. The method of claim 12, wherein analyzing the samplecomponents by multiplexed targeted selected ion monitoring (SIM) togenerate an inclusion list comprises utilizing an ion trap or orbitrapmass analyzer; and wherein performing iterative mass spectral DDAcomprises utilizing the ion trap or orbitrap mass analyzer fitted with asegmented quadrupole mass filter.
 14. The method of claim 12, whereinanalyzing sample components by multiplexed targeted selected ionmonitoring (SIM) to generate an inclusion list comprises segmenting massto ratio window settings in which multiple segments are included andeach segment has multiple windows.
 15. The method of claim 14, whereinmultiple segments is three segments or four segments; and whereinmultiple windows is 10 windows.
 16. The method of claim 14, wherein eachwindow within a segment has the same window width.
 17. The method ofclaim 12, wherein the cell culture medium is a soy-based cell culturemedium and/or is for a recombinant cell-based production system.
 18. Themethod of claim 17, wherein the cell culture medium sample is obtainedfollowing incubation with the recombinant cell-based production system.19. The method of claim 18, wherein the recombinant cell-basedproduction system is a mammalian system.
 20. The method of claim 18,wherein the recombinant cell-based production system is for proteinproduction, wherein the protein is an antibody, a fusion protein, arecombinant protein, or a combination thereof; and wherein the antibodyis a monoclonal antibody of isotype IgG1, IgG2, IgG3, IgG4, or mixedisotype.